Method for the purification of glycero-lipid-splitting enzymes

ABSTRACT

Enzymes acting on glycero-lipids, for example, lipase, phospholipase, lysophospholipase or phosphatidate phosphatase, are purified by the steps of: contacting an aqueous enzyme solution with a carrier which is a fatty acid ester of a waterinsoluble polysaccharide or a derivative thereof containing hydroxyl groups, thereby to immobilize the same, and eluting the enzyme with a solution containing a surface active agent.

United States Patent [1 1 Horiuchi et al.

[451 Aug. 26, 1975 2 l] App]. No; 450,083

[30] Foreign Application Priority Data Mar. l0, [973 Japan i. 48-28425 [52] US. Cl. l95/66 R; 195/66 R [51] Int. Cl. C07G 7/02 [58} Field of Search l95/66 R, 63, 68, DIG. ll

[56] References Cited OTHER PUBLICATIONS Zaborsky, Immobilized Enzymes, Published 1973 by CRC Press, (Division of the Chemical Rubber Co).

Primary Examinerl .ionel M. Shapiro Attorney, Agent, or Firm-Young & Thompson [57] ABSTRACT Enzymes acting on glycero-lipids, for example, lipase, phospholipase, lysophospholipase or phosphatidate phosphatase, are purified by the steps of: contacting an aqueous enzyme solution with a carrier which is a fatty acid ester of a water-insoluble polysaccharide or a derivative thereof containing hydroxyl groups, thereby to immobilize the same, and eluting the enzyme with a solution containing a surface active agent 1 Claim, N0 Drawings METHOD FOR THE PURIFICATION OF GLYCERO-LIPID-SILI'I'TING ENZYMES This invention relates to a novel method for the puri fication of glycero-lipitbsplitting enzymes. i.e. enzymes having substrate specificity for glycerides or glycerophosphatides.

Purification methods for enzymes employing chro matography are knownv Among these, affinity chromatography is the most accurate separation method. However such a method is almost impossible to conduct as a large scale industrial process due to the complicated equipment and expensive carrier required.

As a result of various investigations performed by the present inventors on methods for the purification of enzymes having substrate specificity for glycerides or glyccro-phosphatides. it has now been found that said enzymes can be specifically adsorbed by a carrier which is a fatty acid ester of a water-insoluble polysaccharide or a derivative thereof containing hydroxyl groups. and that the thusadsorbed enzymes are not eluted by any change of pH or increase of ionic strength but can be easily eluted with a liquid containing a surface active agent.

The present invention thus provides a method for the purification of enzymes having substrate specificity for glycero-lipids. which comprises contacting a said enzyme with a carrier which is a fatty acid ester of a \va ter-insoluble polysaccharide or a derivative thereof containing hydroxyl groups. and eluting the said en- /yme with a liquid containing a surface active agent.

An object of the present invention is to increase specific activity ofenzy mes having substrate specificity for glyccro-lipids with superior recovery Another object of the present invention is to provide a superior purification procedure for the enzymes including the purification of isozymcs thereof.

In the present in\ ention. the enzyme having substrate specificity for glycero-lipids is defined as an enzyme which hydrolyzes a lipid of the formula:

wherein R and R are hydrogen or an acyl group. especially a fatty acid residual group. R and R are not both hydrogen at the same time. and R is hydrogen. an acyl group. especially a fatty acid residual group. a residual group of phosphoric acid. phosphryl choline. plt0sphoryl ethanol amine. phosphoryl serine. or phosphoryl inositol. and the said enzyme is generally designated as lipase. phospholipase A. B. C and D. phosphatidate phosphatase. lysophospholipase or the like.

The fatty acid esters of water-insoluble polysaccharides or derivatives thereof containing hydroxyl groups. which are used as carriers for purification of enzymes in accordance with the present invention. are obtained by any usual method such as esterification of waterinsoluble polysaccharides or derivatives thereof con taining hydroxyl groups with reactive derivatives of fatty acids. especially acid halides thereof.

Eyamplcs of the water-insoluble polysaccharides or derivatives thereof containing hydroxyl groups are natural plants fibers such as cotton. linen. jute or Manila hemp; cellulose fibers such as regenerated fibers (e.g.

viscose rayon); cellulose powder; cellulose derivatives such as carboxmethyl-cellulose. phospho-cellulose. sulfomethyl'cellulose. sulfoethylcellulose, panninobcnzyl-cellulose, aminoethyl-cellulose. diethylamino ethyl-cellulose, triethylaminoethyl-cellulose. or guanidinocthyl-cellulose: crosslinked gel of dextranepichlorohydrin (hereinafter designated dextran gel); dextran gel derivatives such as carboxymethyl-dextran gel. diethylaminoethyl-dextran gel or sulfoethyl dextran gel; and agar.

The cellulose derivatives may be chemically synthesized by any ordinary method [refer to J. Am. Chem. 500.. 78. 75l (1956)] or. alternatively, commercially available ion exchangers of cellulose may be used.

The dextran gel may be prepared by known methods from crystalline dextran and epichlorohydrin. (ommercially available dextran gels having various degrees of erosslinking, i.e. SephacleiU (trade name). may also conveniently be used. As dextran gel derivatives. products prepared by carboxymethylation. diethylamino ethylation or sulfoethylation of dextran gels may preferably be used. However commercially available CM- Sephadex. DEAE-Sephadex or SE-Sephadct may also be used.

As fatty acids to be used for esterification. saturated or unsaturated fatty acids having six or more carbon atoms are advantageously used. These fatty acids may have either branched or straight chain structure. A mixture containing several kinds of fatty acids having different carbon drains may also be used.

According to the present invention. the enzyme hav ing substrate specificity for glycero-lipids is treated by merely contacting a solution containing said enzyme with a carrier which is a fatty acid ester of polysaccharide as mentioned above. The treatment. therefore. may be performed batchwise or in a column.

The temperature and pH for that treatment may be varied freely. so long as no detrimental effect on the enzyme activity is caused. And it may preferably be car ried out at 2() to 30C. and at pH 7 to 8.

The fatty acid ester of polysaccharide has poor affinity for water. and therefore it is preferably subjected to pretreatment with hydrophilic organic solvents such as acetone or lower alcohols.

The thus-adsorbed enzyme on a fatty acid ester of said polysaccharide is easily eluted by treating with a liquid containing a surface active agent. after washing.

In the process of the present invention. the surface active agent for eluting enzyme may preferably be selected by its superior eluting activity on the enzyme. so far as it does not denature the enzyme. For example. triton X-IUU (trade name). adekatolc 45-5-8 (trade name). BL-Q-EX (trade name). tween EU (trade name). tween 4t) (trade name), cation FB (trade name cation DTQUS (trade name). or sodium deoxychlolatc may advantageously be used.

The concentration of surface active agent may vary according to the nature of the enzyme and of the surface active agent and it may preferably be more than (Hi3 f (v/v).

In the present invention. fatty acid esters of waterinsoluble polysaccharides or derivatives thereof con taining hydroxyl groups used for the purification of enzyme solutions can be reused by washing with lovver alcohols or acetone after removing the enzyme.

The effect of the surface active agents on enzyme activity and absorbability of the said enzyme to palmitoyl celluloses are measured using phospholipase A. C or D, lysophospholipase and phosphatidic acid phosphatase as the enzyme having a substrate specificity for glycerolipids, together with using palmitoyl cellulose as a car- Phospholipasc A.

A reaction mixture containing purified egg yolk lecithin l umole), NaCl l5 pImoles). CaCl- (2.3 ptmoles). ED'I'A (0.0l 3 umole). ethyl ether (0. l 5 ml.)

rier. The results are shown in Table l. 5 and Tryton X-IUU L5 mg.) per 1.4 ml. was agitated Table l Lysophosphosphatidic acid phospholipase A phospholipase C phospholipasc D pholipase phosphatase surface active agent concentration i" ii i" ii* i ii i* ii i* ii 0.00l V/VZ l26 '49 I 48 0.005 l2? 8) ll I I I l9 63 106 32 Triton X-l00 0.0] [22 iii H2 3 ll) 47 132 I) 003 I00 60 I I3 0 I I5 44 MI I 0.2 I00 33 II5 0 I it 39 I53 0 0.001 V/Wi l26 99 I09 69 0.005 l28 85 I07 29 I00 68 I29 37 Adecatol 45-5-8 0.0I II I 34 I00 0 I27 54 I37 24 0.05 l (is II5 7 I37 27 Ml 9 0.1 125 3 [00 U 142 48 149 0 0.05 v/v'i I05 3| I21 72 I48 4 Tween 20 0.1 I00 23 I03 0 I59 0 0.00I V/V'Z I04 I00 90 6S 0.0I II3 R5 81 4| I 44 I22 90 0.03 I06 25 82 34 I39 39 I47 63 Deoxycholate Na 0.l l05 0 I00 0 0 I33 46 I47 I0 0.2 no 0 I20 4| I47 6 Cation FB [1.] 37

0.003 V/V'Ji I06 83 ll l 97 Cation DT 205 0.0I II5 9 I22 96 0.03 I I3 0 I27 I47 0.I Ho 0 0 128 240 None I00 I00 I00 I00 I00 xu I00 I 00 I00 I 00 i relative activity (Z l ii absorption ratio ('1 In Table l. the assay method is as follows: vigorously with a magnetic stirrer for 5 minutes. Then i. effect of surface active agent on enzyme actvit 45 L4 ml. of the substrate mixture was adjusted to pH 8.0 with 0.0I N NaOH in a titration vessel for automatic The relative ratio of enzyme activity was determined titration recording apparatus. by the ratio of enzyme activity on enzyme solutions [he reaction was initiated by adding ().I ml. of enwith or without surface active agent. cfkct Wrhce dive l em n ma absorb! en zyme solution to the thus-obtained mixture. The reacmt on mini l nu] N g 50 tion rate was determined by titrating the liberated fattv a l I e t .e: p l acids with 0.0I N NaOH In the presence of nitrogen After absorbing theenzyine on palmltoyl cellulose as glm im i t ining the pH at 8.0. a carrier surface active agent was added and stirred. The cnzyme activity Capahh liberating l m y and thei the enzyme solution was separated from the fatty acids per minutc at 0 under he p carrier. lhe activity of enzyme solution separated with "(ms was defined us unit or without adding surface active agent was compared 2 usuphimphoiipusu l down) of thcfmgm'fll cnzynlm To an enzyme solution ((1.l ml.) was added a reacf f f f l l f lmlmc adding f tion mixture (0.9 ml. I containing lysophospbolipid 3.0 lhc m both 'wnv'mis rclimvc Y umoles) and acetate buffer (pH 4.0. 200 ptmoles] and A h n TuhlC ilk HCIiVC llgcm Lh the mixture was incubated at 37C. for 30 minutes. clutes the cnvyme absorbed on palmitoyl cellulose may Aft r adding ethanol (20 ml.) to terminate the reacprcferably selected so as not to inhibit the enzyme acm q d fatty id cstcr was d b h h tivity and so as to have a concentration corresponding d m mcthud I M Augugtyn ct Ana]. to a lower adsorption ratio (i.c. a higher clution ratio). 'hgpn 3L 246 25() 1969) Enzyme activity was de- Activities olthe enzymes having substrate specificity termined by decrease in the amount of ester. The optit'or glycero-lipids are assayed by the following procetlures:

cal absorption ratio of the colorired solution was dctermined by measuring the absorption at 525 mu exactly 5 minutes after adding FeCl; ethanol solution. One amole of decrease in the amount of fatty acid ester per l hour is designated as one unit.

3. Phospholipase C.

A reaction mixture containing egg yolk phosphatide (5.2 pmoles), CaCl- (5 umoles), sodium deoxycholate (300 pg.) and tris-HCl buffer 100 amoles at pH 7.5) per 1.9 ml.. was subjected to supersonic treatment at 9 KC for l minutes. 180 W. To 1.9 ml. of this solution was added 0.l ml. of enzyme solution and incubation was performed at 37C. for 30 minutes.

After 30 minutes. the reaction was terminated by addition of 2.571 lauryl benzene sulfonate (0.9 ml.) and then 0.l ml. of 12'. ml! alkaline phosphatase (40 units: enzyme amount capable of hydrolyzing 40 moles of p-nitrophenyl phosphate at 37C. at pH 7.5 per minute) was added to carry out the reaction at 37C. for 30 minutes. The reaction was terminated by the addition of l ml. of trichloroaeetic acid and the resulting mixture was supplemented with 1 ml. of 5% barium acetate to precipitate lauryl benzene sulfonate and unchanged phosphatide. Each 2 ml. of the filtrate obtained by removing the precipitate with filter paper was sampled in a test tube by centrifugation. and the quantity of inorganic phosphate was determined from the absorption at 720 mp. by the method of Allen modified by Nakamura [refer to .l. Jap. Agr. Chem. Soc, 24. l 1950] I. Before the colorimetric analysis. the precipitate of barium sulfate formed should be removed centrifugally (3000 r.p.m.. minutes). The enzyme unit is the activity liberating l pmole of phosphorylcholine per hour under the above conditions.

4. Phospholipasc D.

a. Preparation of substrate emulsion.

l'en egg yolks were blended in 500 ml. of cold acetone with a whirling blender and thereafter left to stand. The precipitate formed thereby was collected by filtration, washed twice with 200 ml. of cold acetone. and admixed with 300 ml. of chloroform-methanol (1:1) mixture to extract phosphatide. The insoluble portion was separated by filtration.

The extraction procedure with this mixture was repeated twice. The extract was concentrated under reduced pressure until the solvent was removed. whereupon 300 ml. of acetone were added to the concentrate. which was left to stand for 24 hours at l0C. The precipitate formed was collected by filtration. dis solved in 100 ml. ofcthyl ether and stored at l0C. to provide egg yolk phosphatide (I70 moles of phosphatide/ml.

5 moles of this egg yolk phosphatide were mixed with 0.l ml. of ethyl ether. I00 [.LflltllBS of acetate buffer solution (pH 5.5). 200 pmoles of CaCl and deionized water to a total volume of L5 ml. The mixture was subjected to supersonic treatment at 0C. for H) minutes to prepare the substrate emulsion.

b. Procedure.

To 1.5 ml. of the substrate emulsion prepared in the above manner was added 0.5 ml. of enzyme solution. After thorough mixing, 0.5 ml. of ethyl ether was quickly layered. whereby the reaction was carried out at 37C. for minutes. After the reaction was over. 1.0 ml. of trichloroacetic acid was added to the reaction mixture. After the mixture was left to stand at 0C. for [0 minutes, 2 ml. of deionized water was added thereto and the precipitate was removed by filtration. The filtrate was kept at 60C. for l0 minutes to remove ethyl ether. After cooling on ice bath. to 2.0 ml. of the filtrate was added 0.2 ml. of 15.7% l- ,-207( Kl solution. The mixture was left to stand for 20 minutes and then subjected to centrifugal separation (3000 r.p.m.. 10 minutes. at 5C.). The supernatant solution was removed by aspiration by the use ofa capillary tube. The precipitate was dissolved in 10 ml. of l. Z-dichloro ethane. The amount of choline was measured by the increase in absorbance at 365 mg.

The enzyme activity unit is that liberating l umole of choline per hour under the above conditions.

5. Phosphatidate phosphatase.

a. Preparation of substrate emulsion.

One ml. of phosphatidic acid (5] mole/ml.) was put in a supersonic treatment vessel followed by volatilization of the ethyl ether and admixture with 5 ml. of purified water. The mixture was further subjected to supersonic treatment ()KC. 180W) at 0C. for 10 minutes to prepare the substrate emulsion.

b. Procedure for assay of enzyme activity.

The reaction was started by the addition of 0.1 ml. enzyme solution into 0.9 ml. of a reaction mixture containing 0.1 ml. of the phosphatidic acid as described above. 0.5 M acetate buffer solution (0.1 ml. 0.0l M CaCl solution (0.l ml.) and deionized water (0.6 ml. 1. The reaction was carried out at 37C. for 30 minutes. and terminated by the addition of 1.0 ml. of 10% trichloroacetic acid. Then 1.0 ml. of 1% serum albumin solution was added to the mixture and mixed well. Unaltered substrate and protein were removed by centrifugation (3000 r.p.m.. l0 minutes The supernatant solution was analyzed similarly as described in Method 3 hereinbcfore.

The enzyme activity unit was that liberating l amole of inorganic phosphoric acid at 37C. per minute under the above conditions.

6. Lipase.

a. Preparation of emulsion.

One liter of deioniYcd water containing l8.5 g. of a polyvinyl alcohol and [.5 g. of another polyvinyl alcohol was warmed with stirring at to C. for l hour. thereafter cooled and filtered to prepare a PVA solution.

lnto a homogenizer vessel of stainless steel were charged 75 ml. of the PVA solution and 23 g. of olive oil and. after homogenization at 1 L000 r.p.m. at 0C. to 5C. for l0 minutes. PVAolive oil emulsion \vas secured. This emulsion must be used within 24 hours after it has been left to stand at 0 to 5C. for l hour.

b. Method of reaction.

lnto a test tube 24 mm. in diameter and 200 mm. in length were introduced the phosphate buffer solution (2 ml.) and the PVA emulsion (5 ml. After stirring for about 3 seconds with a vibrator. prcincubation was effected at 37C. for 10 minutes. Then. one ml. of the cn zyme solution was added to this mixture and. after stir ring with a vibrator for about 3 seconds. the reaction was initiated. After 20 minutes. the reaction was terminated by addition ol 20 ml. of ethanol-acetone mixture l l:l

The free fatty acids in the resultant reaction mixture were titrated with 0.05 N NaOH. while bubbling the mixture by passing nitrogen gas therethrough. by the use of 3 to 5 drops of I)? alcoholic phenolphthalein as indicator. On the other hand. the reaction mixture without enzyme solution was mixed with 20 ml. of the ethanol-acetone mixture. followed by addition of the Difference in titration values X 2.5 Sample amount In 1 ml. of enzyme solutlon Lipase unit (u) The following examples illustrate the invention.

EXAMPLE l 21) g. of palmitoyl ester of cellulose were immersed inSU); aqueous acetone solution. packed in a column of l .2 X 9 cm. and washed with water. mg. oflyophilized snake venom was dissolved in 60 ml. of H) mM tris-HCl buffer solution (pH 8.0) and centrifuged at 5000 rpm. for 5 minutes. The resulting supernatant solution was charged into the above column packed with palmitoyl ester of cellulose, and passed through at a flow rate of 50 ml./hour. Then the column was washed thoroughly with water and thereafter was eluted with U. l /r Adekaton 45-8-8 and It) mM trisHCl buffer (pH 8.0) at an elution rate of 50 mL/hour to obtain 5 ml. ofeach fraction and the phospholipase A active fractions were collected. The activity ratio ofcrude enzyme to eluted enzyme was assayed by the enzyme activity and amount of protein determined by Lowrys method [1. Biol. Chem. 193. 265 I951 J]. The results were as follows:

Relative Activity (uJmg. protein) lllll lllH Crude Ell/)nh. Solution Elutetl Solution EXAMPLE 2 Example I was repeated. but the palmitoyl ester of gauze was replaced by the following carriers to purify snake venom:

Relative Activity Reeowry tu/m protein) (W;

Ole \l Ester of Cellulose 2-H) 9h fill -Continued Relative Activity Recovery lu/mg. protein) (/r) Palmitoyl Estcr of Sephadcx (1-25 llll ('aproyl Ester of Agar I94 J4 EXAMPLE 3 To portions of finely cut cabbage g.) rich in chlorophyll was added cold deionized water I00 ml.). After stirring by means ofa whirling blender for 5 minutes, the mixture was filtered with gauze. The filtrate was subjected to centrifugation l(),OOO r.p.m.. 20 minutes, at UC.) and the supernatant solution was used as a crude enzyme solution.

Palmitoyl ester of agar (4 g.) was immersed in 507! ethanol. packed in a column of 1.2 X l[) cm. and washed with water. Thereafter cabbage extract as here inabove prepared was passed through the aforesaid column at a flow rate of 60 ml./hr. Then after washing with water. the column was eluted with 0.0571 Adekatol 45- 5-8 and 0.0] M phosphate buffer (pH 7.0) at a flow rate of 50 ml./hr. Eluate was collected in fractions of 10 ml. each thereby to obtain phospholipase D-rich fractions. The relative activities of crude enzyme and eluate were determined by the same procedure as in Example 1:

Relative Activity Recmcry lu/mg. protein) (/2 (rude Enzyme Solution 27 ltIlJ Eluate III) The eaproyl ester of agar as a carrier was prepared as follows:

3 g. of agar powder and l5 ml. of eaproic acid chloride were added to 50 ml. of pyridine-acetone mixture (lzl After the reaction proceded at 30C. with stir ring for It) hours. the reaction product was washed twice with 50 ml. of ethanol and dried to obtain 3.9 g. of eaproyl ester of agar.

EXAMPLE 4 In Example 3. eaproyl ester of agar was replaced by the following carriers to purify the cabbage extract:

Bacillus cereus IAMI 208 was inoculated to l()() ml. of a liquid culture medium (pH 7.0) containing peptone (1 g. meat extract l g.) and NaCl (0.5 g.) and shake cultured at 30C. for 20 hours. After 20 hours cultivation. the culture broth was subjected to centrifugal separation (SUUO r.p.m.. 20 minutes. at 0C.) to remove bacterial cells. The resulting supernatant solution was used as a crude phospholipase C solution.

3.0 g. of palmitoyl ester of cellulose were immersed in 509 ethanol. packed in a column of l2 X 10 cm. and washed with 0.0l M tris-HCI buffer (pH 7.5 J. The crude enzyme solution (200 ml.) as hereinahove produced was passed through the column at a rate of I50 ml./hr.

Then after washing with 0.0] M-tris-HCI buffer solution the column was eluted with 0.03% Adekatol 45-S- 8 and 0.01 M-tris-HCl buffer solution (pH 7.5) at a flow rate of 150 inf/hr. to collect fractions of 15 ml. each. The obtained fractions showed phospholipase C activity. The relative activities of the crude enzyme solution and of the eluate were assayed by the method described in Example I. as follows:

Rclathe Actnity Recovery (ti/mg. protein) (it ('rude l'n/ymc Solution (15 I l'luate i970 104 Rclatnc Actiut in mg protein) Recmeiy EXAMPLE 7 lt'm'tiflimn )mluiimi lFO 4640 was inoculated into 100 ml. of a liquid culture medium (pH 5.4) containing corn steep liquor 3.5 g. lactose (5.5 g. J. soy hean oil (025 g). Ca((1 (l.0 g.) and KH PO, (0.7 g.) and shake cultured at 26C. for days. After cultivation. mycelial cake was obtained by filtration and washed with deionized water. Then to 5.0 ml. of deionized water was added l g. (wet weight) of mycelia. and this was crushed in a whirling blender for it] minutes at ambient temperature (l2 000 r.p.m.). The mycelial homogcnate was adjusted to pH 7.0 and filtered. The filtrate was centrifuged at l0 000 r.p.m. for 10 minutes at 0"('. to obtain the supernatant liquid showing lysophospholipase activity.

2 g. of oleoyl ester of gauze were immersed in 50'): ethanol. packed in a column of 1.2 X 9 cm. and washed with 0.0] M acetate buffer solution (pH 50) containing 1 M NaCl. A solution ml.) of the same buffer containing 5 ml. of the mycelial extract was passed through the column at a How rate of 150 ml./hr. After washing with the same huffer solution. the enzyme was eluted with 0.01% sodium t'leoxycholate and 0.0] M phosphate buffer (pH 7.0). and fractions off) ml. each gathered. The lysophospholipase-active fractions were assayed:

EXAMPLE 8 ln example 7. oleoyl ester of gauze was replaced by the following carriers:

.3implomycrav niimbilix was inoculated into a liquid medium (pH 05ml.) containing peptone (l g.) glucose (0.5 g.) corn starch (0.5 g) NaCl (0.3 g.) and MgSO (0.05 g.), shake cultured at 26C. for 3 days and the culture broth was filtered. To the filtrate ml.) were added barium acetate lb g.) and NaCl (2.4 g. The precipitate thereby formed was removed centrifugally (5000 r.p m., 10 minutes) and the supernatant solution was provided as crude phosphatirlate phosphatase solution (0.18 /ml.)

Palmitoyl ester of cellulose (15 g.) was immersed in 50% ethanol. packed in a column of 1.2 X cm. and then washed thoroughly with deionized water. into the column was fed the crude phosphatide phosphatase solution (80 ml.) as prepared hereinahove at a flow rate of I50 ml./hr. Then the column was washed successively with I59? NaCl solution (l0 ml.). 075% NaCl solution (l0 ml.) and ().l. NaCl solution (50 ml.). After washing. the enzyme was eluted with 0.1% Adekatol 45-5-8 at a flow rate of 150 ml./hr. and the thus-obtained eluate was fractionated into fractions of l5 ml. each. Phosphatidate phosphatase active fractions were collected and assayed as follows:

EXAMPLE l0 Palmitoyl ester of gauze 35 g.) was immersed in 50% aqueous acetone solution. packed in a column and washed with water. Then an aqueous solution of lipase (250 ml. 75 u/mf, 40 IL/111g. protein) was passed through the column, followed again by washing with water (1000 ml.). The enzyme was eluted with 0.3)? Adekatol 45-5-8 ml.) The eluate was concentrated to 20 ml. in vacuo in the presence of silicone oil as an antiform agent. thereafter acetone (30 ml.) was added to precipitate the material. The precipitate was centrifugally removed and to the supernatant was further added acetone (50 ml.). The precipitate formed thereby was collected. dissolved in 10 ml. of water and then freeze dried to obtain 24 mg. of lipase powder (potency: 400 jJ/n'lg. protein).

Having described our invention. we claim:

1. A method for the purification of an enzyme having substrate specificity to glycero-lipids selected from the group consisting of glycerides and glycerophosphatides, which comprises the steps of:

contacting an aqueous solution of a said enzyme with said fatty acid having a! least (1 carbon atoms. and

a carrier selected from the group consisting of fatty eluting the adsorbed enzyme with an aqueous solu acid esters of water-insoluble polysaccharides and tion of a surface active agent. derivatives thereof containing hydroxyl groups. 

1. A METHOD FOR THE PURIFICATION OF AN ENZYME HAVING CUBSTRATE SPECIFICITY TO GLYCERO-LIPIDS SELECTED FROM THE GROUP CONSISTING OF GLYCERIDES AND GLYCERO-PHOSPHATIDES, WHICH COMPRISES THE STEPS OF: CONTACTING AN AQUEOUS SOLUTION OF A SAID ENZYME WITH A CARRIER SELECTED FROM THE GROUP CONSISTING OF FATTY ACID ESTERS OF WATER-INSOLUBLE POLYSACCHARIDES AND DERIVATIVES THEREOF CONTAINING HYDROXYL GROUPS, SAID FATTY ACID HAVING AT LEAST 6 CARBON ATOMS AND ELUTING THE ADSORBED ENZYME WITH AN AQUEOUS SOLUTION OF A SURFACE ACTIVE AGENT. 